Current studies indicate that immune protection against cancer requires the generation of a potent cellular immune response against a unique tumor antigen expressed by a malignant cell. Thus, successful immune protection would first require identifying a unique antigen in the tumor cells (tumor specific antigen) and then inducing a potent T-cell response targeted to the tumor antigen. These tumor-associated antigens, however, would still be recognized by immune cells as ‘self’ molecules, and so no true activation of the immune system would occur. Thus, two obstacles in targeting these tumor-associated molecules as a vaccine include the unresponsiveness of the immune system to ‘self’ molecules, which restricts its ability to generate potent cellular immune responses, and preventing the generated immune response from being directed to normal cells that express the target antigen.
Proteins that show promise in overcoming these problems include heat shock proteins (HSPs). HSPs include a collection of ubiquitously expressed cytoprotective proteins, which are expressed by cells under conditions of cell stress, such as increased temperature, viral infection and oxidative stress. Certain HSPs have been shown to have immunomodulatory effects, such as the induction of cytokines and the promotion of cell activation and maturation (see, Pockley A G, Lancet 363 (9382) 469-476 (2003)).
Gp96 is an HSP of particular interest. Gp96 is a 96 kDa glycoprotein localized to the endoplasmic reticulum, which can also be found at the cell surface. Gp96 has been shown to be released into the extra cellular space during necrotic cell death and activates dendrite cells and macrophages by realizing inflammatory cytokines and inducing dendrites cells to mature. Extra-cellular gp96 has been known to activate dendrite and macrophages by modulating inflammatory cytokines and inducing maturation of dendrites.
Gp96 has the ability to transfer antigenic peptides for their MHC-class 1-restricted presentation and allows gp96 to function as an efficient messaging system alerting the immune system of an infection. This includes the receptor-mediated uptake of gp96 by dendrite cells. The receptor is CD91, which is known as the α2 macroglobulin (α2M) receptor expressed on phagocytes. The presentation of gp96-associated peptide by antigen-presenting cells (“APC's”) is induced by α2 macroglobulin. Gp96 is bound by CD91 on dendrite cells and internalized. Gp96 induces the expression of co-stimulatory molecules and the release of interleukin 12 (IL-12) and tumor necrotic factor α (“TNFα”) by the APC.
Certain infections, such as by the human immunodeficiency virus, have also presented challenges in targeting the disease-causing organism and neutralizing it. Typically, infection with the human immunodeficiency virus, HIV-1, eventually causes acquired immunodeficiency syndrome (AIDS) and an associated syndrome, AIDS-related complex (ARC). Neutralizing this virus has proved difficult, largely because its structure obstructs immune system access to viral epitopes and its genetic material is highly variable. Accordingly, researchers have been seeking prophylactic and therapeutic methods for preventing or controlling HIV which are not dependent upon antibody-mediated immunity.
It has been recognized that denying entry into CD4+ cells to the HIV-1 virus could at least slow the progress of the infection and alleviate, if not cure, the disease and/or its symptoms. The complex mechanism by which the virus crosses the cell membrane has been widely investigated. Broadly, the entry of human immunodeficiency virus into, for example, CD4+ Th1 cells (T-helper type 1 cells), is dependent upon a sequential interaction of the gp120/gp41 subunits of the viral envelope glycoprotein gp160 with the CD4+Th1 cell surface glycoprotein and the cell surface receptor CCR5. The “env” gene of HIV encodes a single protein, gp160. Gp160 travels to the cell surface where cellular enzymes cleave it into gp120 and gp41. If and when new virus particues bud off from the host cell, these two pieces lie on opposite sides of the virus membrane. Gp120 sits on the outside of the virus particle, forming the virus' spikes, while gp41 sits just on the inside of the membrane, with each gp41 unit being anchored to a gp120 through the membrane. On binding of gp120 with its cell surface binding sites, a conformational change in the latent gp41 subunit through an intermediate state to an active state is initiated, inducing fusion of the viral and cellular membranes and transport of the virus into the cell (Weissenhom et al., Nature, 387:426-30 (1997)).
Numerous binding experiments have been conducted in an effort to find antiviral ligands that will effectively compete with the HIV-1 for CD4+ gp and/or CCR5 binding sites, or that will preferentially block gp120 and/or gp41 binding domains. The frequent mutability of the gp41 and gp120 surface antigens, however, lessens the chance that traditional approaches for creating immunity will be successful against HIV. Therefore, new approaches employing gp41 and gp120 complexes with new immunomodulators which increase their immunogenicity are needed.